Calcofluor which is represented by the following structural formula is a fluorescent compound which specifically binds to .beta.-glucan to have an increased degree of fluorescent intensity due to the binding. Jorgensen et al of Carlsberg Co. in Denmark have reported a flow injection method using this compound (Carlsberg Res. Commun., Vol. 53, pp.277-285, 1988; Analytica-EBC, 3.11.2). ##STR1##
Some other researchers have also reported a flow injection method using calcofluor on the basis of the same principle (Journal of the Institute of Brewing, Vol. 95, p.327, 1989; Journal of the Institute of Brewing, Vol.93, p.396, 1987). Recently, commercial flow injection system using calcofluor have been marketed by Tecator Co. in Sweden and by Fiatron Co. in USA (Journal of American Society of Brewing Chemists, Vol. 93, p.396, 1987).
These are all applied system from the system shown in FIG. 1 or FIG. 2, in which a flow of a sample or a solution containing a sample is mixed with a flow of a reagent solution prepared by dissolving .beta..about.35 mg/liter of calcofluor in a tris or glycine buffer (pH 8 to 10) by which calcofluor is bound to .beta.-glucan in the sample, using a suitable tube, and the increase in the fluorescent intensity of the thus-bonded compound is measured using a fluorescence detector.
To determine the .beta.-glucan content in the sample such as wort, beer, etc. by these methods, a solution containing a known concentration of purified .beta.-glucan extracted from barley is used as a standard.
However, such conventional flow injection methods using calcofluor were reported to involve fluctuation of the measured values due to the effects of sugars in the sample wort or ethanol in beer which also vary depending on the measurement conditions such as sample injection volume and empty volume of the mixing zone, resulting in variation in .beta.-glucan content even in an identical sample.
In addition, lower molecule components (saccharides, ethanol, etc.) having inhibitory effect on fluorescent reaction of calcofluor were found recently to be present in wort or beer, and the contents of such components in wort or beer were found to vary depending on the species and amount of the malts employed.
It is obvious that the above-mentioned substances hindering fluorescent reaction of calcofluor such as saccharides, ethanol, etc. cause errors in the measurement of the .beta.-glucan content in wort or beer by the conventional flow injection methods using calcofluor.
Accordingly, to establish a system which cannot be affected by saccharides, ethanol and hindering substances of calcofluor fluorescent reaction present in a sample to be measured is essential for the purpose of improving the accuracy of .beta.-glucan measurement, and such system is desired to be developed.
Thus, an objective of the present invention is to improve the accuracy and reproducibility of the measurement of .beta.-glucan by the flow injection methods using calcofluor, by means of establishing the system and measurement condition in which a short gel filtration column is employed to allow high molecular .beta.-glucan having a molecular weight of 10,000 or higher to be passed through without being retained in the column support while low molecular saccharides, ethanol and substances hindering fluorescent reaction of calcofluor are retained by gel particles as column support for a period of several seconds to several ten seconds, whereby achieving elution as separated from .beta.-glucan.
In addition, optimization of the column size to achieve an analysis time equivalent to that in the analysis without such column (several minutes (preferably 3 minutes) or shorter) is another objective of the present invention.
A method for measuring .beta.-glucan by a flow injection system using calcofluor according to the present invention employs a method wherein a sample flow is passed through a gel filtration column by which it is possible that after injecting the sample into the flow of an eluent an analysis target, namely, high molecular .beta.-glucan having a molecular weight of 10,000 or higher is eluted within a certain period (within several minutes (preferably 3 minutes)) while the elution of low molecular components such as maltose, ethanol and substances hindering fluorescent reaction of calcofluor is retarded in relation to the elution of high molecular .beta.-glucan, whereby separating .beta.-glucan from the components causing fluctuation of the measured values, and the sample thus having been passed through the column was then measured by a fluorescent detector.
As mentioned above, the present invention is a method for measuring .beta.-glucan wherein a gel filtration column is placed between the sample injection port and the detector in the system, said column being characterized in that the volume of the interstice outside of the gel particles (void volume) is not larger than the volume of the fluid eluted from the column within a certain time period (preferably 3 minutes) and the column content volume (the volume of the interstice outside of the gel particles plus the volume of the solvent within the gel particles (inner volume)) corresponding to the volume of the eluent with which the low molecular substance migrating into the network of the gel particles are eluted from the column is 10 times or more as large as the sample injection volume, whereby achieving that the analysis target high molecular .beta.-glucan does not migrate into the network of the gel particles but can readily be eluted from the column when the eluent in the volume equal to the total volume of the interstice of the gel particles is passed through the interstice of the gel particles.
When the column content volume mentioned above is smaller than the sample injection volume, satisfactory separation between high molecular .beta.-glucan having a molecular weight of 10,000 or higher and low molecular saccharides, ethanol and substances hindering fluorescent reaction of calcofluor can not be achieved.